Ox40r binding agents

ABSTRACT

The present invention discloses peptides isolated from the extracellular domain of OX40 Ligand (OX40L) capable of binding OX40 Receptor (OX40R) and inhibiting OX40R-OX40L interaction. Such peptides, fusion proteins comprising them, as well as peptides and other molecules designed on their sequences, can be used as OX40R binding agents competing with natural OX40L for blocking OX40R-mediated cell signaling in the prophylaxis and/or treatment of diseases related to activated T cells.

FIELD OF THE INVENTlON

The present invention concerns novel peptides capable of modulating OX40Receptor-OX40 Ligand interactions.

BACKGROUND OF THE INVENTION

The cell regulatory system constituted by the membrane proteins OX40Receptor (indicated in the literature also as OX40R, OX-40, OX-40Antigen, TNFRSF4, or CD134) and OX40 Ligand (indicated in the literaturealso as OX40L, glycoprotein gp34, ACT-4-L, TNFSF4, CD134 Ligand, orCD134L) has a prominent role in the regulation of immune responses aswell as in the formation of secondary lymphoid tissue, similarly toother proteins belonging to the tumor necrosis factor ligand/receptorsuperfamilies (Gravestein L and Borst J, 1998; Weinberg A, 2002). Manyevidences on these activities have been provided by clinicalobservations and by animal models, for example by gene targetingexperiments (Chen A et al., 1999; Kopf M et al., 1999; Murata K et al.,2000).

OX40 Receptor (OX40R, from now on) is a cell surface antigen, member ofTNFR family, transiently expressed following T cell receptor (TCR)engagement and acting as a co-stimulatory receptor. It is considered asa highly specific CD4⁺ or CD8⁺ activation marker for T cells, beingoften over-expressed in inflammation sites associated to immunologicalpathologies, such as in multiple sclerosis or rheumatoid arthritis, aswell as in tumor-infiltrating lymphocytes and in the peripheral blood ofanimal models of graft-versus host disease.

OX40 Ligand (OX40L, from now on) is a transmembrane protein, originallyidentified as a protein stimulated by human T cell lymphotropic virus 1infection and CD40 activation (Miura S et al., 1991), having structuralsimilarity to TNF and capable of forming cell-bound or secreted trimers.It is present on activated, antigen-presenting B and T cells, as well asdendritic cells, vascular endothelium cells and other non-hematopoietictissues (for example heart, skeletal muscle, and pancreas).

OX40L interacts with OX40R as a homotrimer with a high affinity(Kd=0.2-0.4 nM), and various binding assays have been tested on thissystem (Taylor L et al, 2002; Taylor L and Schwartz H, 2001;Al-Shamkhani A et al., 1997). However, no tridimensional structure hasbeen solved so far, neither detailed structure-activity studies havebeen performed, in order to provide any further molecular details on themechanism of OX40L-OX40R interaction.

The interaction between OX40L and OX40R has a co-stimulatory effect toOX40R-expressing effector T cells, leading to a more robust cellresponses due to the up-regulation of the cytokine production by Thelper cells (Th1 and Th2) and to an increased survival of memory Tcells through the inhibition of activation-induced cell death.Confirming evidences were also obtained in the transgenic mice lacking afunctional OX40L gene and in autoimmunity animal models, where it wasdemonstrated that blocking the OX40R-OX40L interaction or depletingOX40R-positive T cells reduces clinical signs of autoimmunity.

Moreover, OX40L induces, upon OX40R binding, the expression of severalgenes, including the C-C chemokine RANTES, confirming the resultsobtained in endothelium models where OX40R-OX40L system appears involvedin the control of activated T cells extravasation (Kotani A et al.,2002).

Cumulatively, these expression and functional data raise the possibilitythat the signal transduction pathways regulated by OX40L-OX40Rinteractions may help to prolong antigen-specific proliferativeresponses or otherwise influence the persistence, differentiation orreactivation of effector/memory T cell populations.

The interest on OX40R-OX40L system is related to the fact that, even ifthe intracellular signaling mechanisms have not yet completelyunderstood, the expression profile of OX40R makes this protein apeculiar target for CD4 T cells mediated diseases in clinical settings,for example in multiple sclerosis, where it is necessary to deleteauto-reactive T cells. The hypothesis is that the products modulatingOX40R activity may not have the serious side effects of conventionalimmunosuppressive therapies for autoimmune diseases and transplantrejection, which target all T cells.

The therapeutic potential of modulating the interaction between OX40Land OX40R was recognized by in vivo generated results obtained withOX40L-targeted immunotoxins (Weinberg A et al., 1996), anti-OX40Rantibodies (Bansal-Pakala P et al., 2001), anti-OX40L antibodies (StuberE and Strober W, 1996; Yoshioka Y et al., 2000; Tsukada N et al., 2000),and OX40L-lg fusion proteins (Higgins L M et al., 1999; Weinberg A etal., 1999). These compounds are intended either to antagonizeOX40L-OX40R interaction (for preventing the accumulation of activatedCD4⁺ T cells at inflammatory sites) or to activate OX40R (as in someother pathological conditions, such as cancer).

Various OX40R binding agent, being either agonist or antagonist ofOX40R, have been disclosed in the prior art as having positive effectson immunization and cancer treatment (WO 95/21915; WO 95/21251; EP978287; WO 99/42585; WO 02/66044; U.S. Pat. No. 6,312,700). However,only large molecule such as the OX40L whole extracellular domain orantibodies are actually disclosed as being effective OX40R bindingagents. This is also due to the fact that no real structure-activitystudies have been performed to characterize this interaction, neitherreliable information can be inferred from the analysis of other TNF/TNFRprotein structures (Bodmer J L et al., 2002).

Since known OX40R binding agents proved to be useful as therapeutic anddiagnostic agents, it would be desirable to identify compounds which,maintaining the binding and OX40L-competing properties of the largemolecules above mentioned, are easier to generate and formulate such aspeptides or other small molecules.

SUMMARY OF THE INVENTION

It has now been discovered that specific peptides derived from theextracellular domain of OX40L can be used as OX40R binding agents. Morespecifically, it has been found that a peptide corresponding to aminoacids 94-124 of human OX40L, as well as fragments of this peptidecomprising amino acids 107-116 of human OX40L, interact with human OX40Rwith high affinity, as shown by two different reliable screeningtechnologies. Such peptides, heterologous proteins comprising theirsequences, as well as peptides and other molecules designed on theirsequences, can be used as OX40R binding agents competing with naturalOX40L for different therapeutic uses. Other features and advantages ofthe invention will be apparent from the following detailed description.

DESCRIPTION OF THE FIGURES

FIG. 1: experimental design of the Alphascreen™ assay initiallydeveloped for demonstrating the feasibility of this approach forstudying OX40L-OX40R interaction.

FIG. 2: dose-dependent binding of OX40L-CD8 to OX40R-IgG₁ as detectedusing the Alphascreen™ assay. (A) Increasing concentrations of OX40L-CD8were incubated with OX40R-IgG₁ at the concentration of 5 nM (◯) or 10 nM(●) for 30 minutes. Biotinylated anti-CD8 antibody (10 nM) was thenadded and 30 minutes later, streptavidin donor (20 mg/ml) and protein Aacceptor (20 mg/ml) beads were added. One hour after these additions,the plate was counted on a Fusion™ reader. Only a very weak signal wasdetected in the absence of OX40R-IgG₁ (▪). (B) Effect of increasingOX40R-IgG₁ concentration on Signal/Background (S/B) ratio. The S/B ratiowas calculated by dividing counts obtained in the presence of OX40L-CD8(100 nM) by background counts (in the absence of OX40L-CD8). Data areexpressed as mean±SEM (Standard Error of the Mean) and all experimentswere performed 3 times in triplicate.

FIG. 3: K_(D) determination of OX40L-CD8 for OX40R-IgG₁. OX40R-IgG₁ (10nM) was incubated in the presence of increasing concentrations ofOX40L-CD8. Specific binding (□) was calculated as the difference betweentotal binding (▪) and non-specific binding (●) determined in thepresence of Suramin (1 mM). Data are expressed as mean±SEM from 3experiments performed in triplicate.

FIG. 4: Competition for OX40L-CD8 binding by OX40L and suramin. OX40-IgG(10 nM), OX40L-CD8 (40 nM) and increasing concentrations of solubleOX40L (●) or suramin (◯) were incubated for 30 minutes prior to theaddition of biotinylated anti-CD8 antibody (10 nM). Thirty minutes laterstreptavidin donor and protein A acceptor beads were added. The graphrepresents the percentage inhibition of the maximal Alphascreen™ signal(obtained in the absence of any inhibitor). Data are expressed asmean±SEM from 3 experiments performed in triplicate

FIG. 5: binding of OX40L fragments to OX40R-IgG₁. (A) sequence of humanOX40L (SWISSPROT Acc. No. P23510), with the indication of the positionof main protein domains (on the top of the sequence), together with thesequences corresponding to the peptides tested by the Alphascreen™ basedcompetition assay or the mouse OX40L-derived peptides P-OX-1 and P-OX-2(on the bottom of the sequence; non-identical amino acids are isindicated). (B) inhibition of binding of OX40R-IgG₁ to OX40L-CD8 by thepeptides P4, P5, and a control without any peptide.

FIG. 6: binding of P5-derived peptides to OX40R-IgG₁ (A) sequence ofpeptide P5, aligned with mouse OX40L-derived peptide P-OX-1 (Stuber Eand Strober W, 1996; indicates non conservative substitution and .indicates conservative substitutions, as indicated in Table I) and withthe sequence of the P5-derived peptides tested with theAlphascreen™-based competition assay. The peptide P5-1a is the boxedsequence in P5-1. (B) Inhibition of binding of OX40R-IgG₁ to OX40L-CD8by the peptide P5-1 in the Alphascreen™-based competition assay,compared with the effect provided by other fragments of peptide P5.

FIG. 7: OX40R-IgG₁ interaction with peptides P5 (A) and P5-1 (B), asmeasured by using fluorescence quenching spectroscopy. Non-linearregression analysis of OX40R-IgG₁ fluorescence changes reveals asaturable binding with the dissociation constant for theOX40R-IgG₁—peptide complex indicated as Kd value for the two peptides.

DETAILED DESCRIPTION OF THE INVENTION

In view of the evidences disclosed in the prior art, there is noindication of a specific peptide sequence into OX40L extracellulardomain that would be useful as OX40R binding agent for inhibitingOX40R-OX40L interaction.

By screening series of peptides derived from OX40L extracellular domain,short amino acid sequences that interact with OX40R with high affinityand compete with OX40L, were surprisingly identified and characterizedas inhibitors of OX40R-OX40L interaction.

Accordingly, the present invention discloses a novel OX40R binding agentwhich is the peptide sequence corresponding to amino acids 94-124 (P5;SEQ ID NO: 6) of human OX40L.

The present invention discloses OX40R binding agents which are peptidesequences of human OX40L consisting of a peptide sequence correspondingto amino acids 94-124 (peptide P5; SEQ ID NO: 6) wherein one or moreamino acids have been deleted, and comprising amino acids 107-111(peptide P5-1a; SEQ ID NO: 13) of human OX40L. In particular thesepeptides have between 5 and 10 amino acids, and, more in particular,have the sequence corresponding to amino acids 107-116 (peptide P5-1;SEQ NO ID: 8), or 107-111 (peptide P5-1a; SEQ ID NO: 13) of human OX40L.

The peptide P5, as well as the P5 fragments above defined, has beenshown (or inferred) to bind human OX40R protein in the examples of thepresent patent application. This binding activity has been tested usingin vitro assays employing recombinant forms of OX40L and OX40R, anddemonstrating that OX40L can be effectively competed by the claimedpeptide sequences, Novel means for inhibiting undesirable OX40R-OX40L.interactions and cell signaling associated to human diseases aretherefore provided by the present Invention.

Fragments of the extracellular domain of human ACT-4-L, which is analternative name of OX40L, were disclosed as potential OX40R bindingagents being associated to functional or structural domains in theextracellular domain of OX40L (WO 95/21915). However, no evidence hasbeen provided in the literature on functional or structural domains ofhuman OX40L corresponding to peptide P5, or to its fragments such as thepeptides P5-1 and P5-1a, as having competing activities towards OX40L.Peptides designed on the mouse OX40L extracellular domain sequence havebeen used as antigens for generating anti-OX40L antibodies (Stuber E andStrober W, 1996), but, also in view of the limited conservation of mouseand human OX40L in this region (FIGS. 5A and 6A), it cannot be inferredthat any of these sequences can efficiently compete human OX40L for thebinding to human OX40R.

The present patent application successfully demonstrates that, even ifno functional or structural domain can be inequivocally predicted bycomparing other ligand-receptor pairs belonging to the same proteinfamilies (Bodmer J L et al., 2002), or by using well-known algorithmsfor protein structure prediction such as PREDATOR, PHD or HNN(accessiible, for example, athttp://npsa-pbil.ibcp.fr/cgl-bin/npsa_automat.pl?page=/NPSA/npsa_server.html), such peptides are surprisingly effective asOX40R binding agents capable of inhibiting OX40R-OX40L interaction.

The term “peptide” is ordinarily applied to a polypeptidic chaincontaining from 4 to 40 or more contiguous amino acids, usually from 4to 20 contiguous amino acids. Such peptides can be generated by methodsknown to those skilled in the art, including partial proteolyticcleavage of a larger protein, chemical synthesis, or geneticengineering.

The term “active” defines the compound showing the OX40R bindingproperties demonstrated for the peptides of the present invention.

The properties of the peptide P5 and of its specific fragmentsexemplified by peptides P5-1 and P5-1a can be maintained, or evenpotentiated, in their active mutants. This category of moleculesincludes analogs of these sequences wherein one or more amino acidresidues have been conservatively substituted, provided they display thesame biological activity characterized in the present invention atcomparable or even higher levels, as determined by means known in theart or disclosed in the Examples below.

In accordance with the present invention, preferred changes in theseactive mutants are commonly known as “conservative” or “safe”substitutions. Conservative amino acid substitutions are those withamino acids having sufficiently similar chemical properties, in order topreserve the structure and the biological function of the molecule. Itis clear that insertions and deletions of amino acids may also be madein the above defined sequences without altering their function,particularly if the insertions or deletions only involve a few aminoacids, e.g., under ten, and preferably under three, and do not remove ordisplace amino acids which are critical to the functional conformationof a protein or a peptide.

The literature provide many models on which the selection ofconservative amino acids substitutions can be performed on the basis ofstatistical and physico-chemical studies on the sequence and/or thestructure of natural protein (Rogov S I and Nekrasov A N, 2001). Proteindesign experiments have shown that the use of specific subsets of aminoacids can produce foldable and active proteins, helping in theclassification of amino acid “synonymous” substitutions which can bemore easily accommodated in protein structure (Murphy L R et al., 2000).The synonymous amino acid groups and more preferred synonymous groupsare those defined in Table I.

These alternative compounds are intended to comprehend molecules withchanges to the selected sequences of human OX40L which do not affect thebasic characteristics disclosed in the present patent application,particularly insofar as its ability of binding and inhibiting OX40R isconcerned. Similar compounds may result from conventional site-directedmutagenesis technique of the encoding DNA, from combinatorialtechnologies at the level of encoding DNA sequence (such as DNAshuffling, phage display/selection) or of amino acids, fromcomputer-aided design studies, or any other known technique suitablethereof, which provide a finite set of substantially correspondingmutated peptides which can be routinely obtained and tested by one ofordinary skill in the art using the teachings presented in the prior artand in the Examples of the present patent application.

The present patent application discloses novel OX40 binding agents beingfusion polypeptides or peptides comprising the amino acid sequence P5,P5-1, P5-1a, or any of their active mutants as defined above, and anamino acid sequence belonging to a protein sequence other than humanOX40L. This heterologous latter sequence should provide additionalproperties without considerably impairing OX40R binding activity.Examples of such additional properties are an easier purificationprocedure, a longer lasting half-life in body fluids, or extracellularlocalization. This latter feature is of particular importance fordefining a specific group of fusion or chimeric proteins included in theabove definition since it allows the peptides characterized as OX40Rbinding agent in this patent application to be localized in the spacewhere not only where the isolation and purification of these peptides isfacilitated, but also where OX40L and OX40R naturally interact.

Additional protein sequences which can be comprised in fusion proteinsincluding the OX40R binding agent of the Invention can be chosen amongstmembrane-bound proteins, extracellular domains of membrane-boundprotein, immunoglobulin constant region, multimerization domains,extracellular proteins, signal peptide-containing proteins, exportsignal-containing proteins.

The choice of one or more of these sequences to be fused to the OX40binding agent is functional to specific use of said agent. As a generalprocedure, these fusion proteins can be produced by generating nucleicacid segments encoding them, using common genetic engineeringtechniques, and cloning in replicable vector of viral or plasmid originwhich are used to modify a Prokaryotic or Eukaryotic host cell, usingepisomal or non-/homologously integrated vectors, as well astransformation-, infection-, or transfection-based technologies. Thesevectors should allow the expression of the fusion protein including theOX40R binding agent in the prokaryotic or eukaryotic host cell under thecontrol of their own transcriptional initiation/termination regulatorysequences, which are chosen to be constitutively active or inducible insaid cell. A cell line substantially enriched in such cells can be thenisolated to provide a stable cell line. In particular, whenever thecells modified to express the OX40R binding agents of the invention aredirectly used or administered, preferred cells are human cells normallyexpressing OX40L, in particular human B cells.

When the additional protein sequence, as in the case of the sequence ofextracellular, export signal, or signal-peptide containing proteins,allows the OX40R binding domain to be secreted in the extracellularspace, the agent can be more easily collected and purified from culturedcells in view of further processing or, alternatively, the cells can bedirectly used or administered.

When the additional protein, as in the case of the sequence ofmembrane-bound proteins, allows the immobilization of the OX40R bindingagent on the surface of the cell, the agent can be less easily collectedand purified from the cultured cells in view of further processing butthe cells can be directly used or administered providing the agent in aform corresponding to the one of natural OX40L, possibly improving itsproperties.

Finally, since OX40L-OX40R interaction is known to involvemultimerization of the proteins, in particular the trimerization (AlShamkhani A et al., 1997). Therefore, the fusion protein may alsoinclude sequence allowing the multimerization of the resulting protein,such as the immunoglobulin constant regions, extracellular domains ofmembrane-bound proteins, or trimerization domains known in the art asbeing present in TNFR-like (WO 00/39295) or in other proteins (WO01/49866, WO 99/10510, WO 01/98507). Other useful protein sequences thatcan be included are the ones providing means of purification by affinitychromatography (Constans A, 2002; Lowe C R et al., 2001).

The polypeptides and the peptides of the present invention can be inalternative forms which can be preferred according to the desired methodof use and/or production, for example as active fractions, precursors,salts, or derivatives.

The term “fraction” refers to any fragment of the polypeptidic chain ofthe compound itself, alone or in combination with related molecules orresidues bound to it, for example residues of sugars or phosphates, oraggregates of the original polypeptide or peptide. Such molecules canresult also from other modifications which do not normally alter primarysequence, for example in vivo or in vitro chemical derivativization ofpeptides (acetylation or carboxylation), those made by modifying thepattern of phosphorylation (introduction of phosphotyrosine,phosphoserine, or phosphothreonine residues) or glycosylation (byexposing the peptide to enzymes which affect glycosylation e.g.,mammalian glycosylating or deglycosylating enzymes) of a peptide duringits synthesis and processing or in further processing steps. Forexample, P5 and P5-1 contain a potential glycosylation site (amino acids114-116 in human OX40L) and this can be modified accordingly during therecombinant expression in the host cell or during chemical synthesis.

The “precursors” are compounds which can be converted into the compoundsof present invention by metabolic and enzymatic processing prior orafter the administration to the cells or to the body.

The term “salts” herein refers to both salts of carboxyl groups and toacid addition salts of amino groups of the peptides, polypeptides, oranalogs thereof, of the present invention. Salts of a carboxyl group maybe formed by means known in the art and include inorganic salts, forexample, sodium, calcium, ammonium, ferric or zinc salts, and the like,and salts with organic bases as those formed, for example, with amines,such as triethanolamine, arginine or lysine, piperidine, procaine andthe like. Acid addition salts include, for example, salts with mineralacids such as, for example, hydrochloric acid or sulfuric acid, andsalts with organic acids such as, for example, acetic acid or oxalicacid. Any of such salts should have substantially similar activity tothe peptides and polypeptides of the invention or their analogs.

The term “derivatives” as herein used refers to derivatives which can beprepared from the functional groups present on the lateral chains of theamino acid moieties or on the N-/ or C-terminal groups according toknown methods. Such derivatives include for example esters or aliphaticamides of the carboxyl-groups and N-acyl derivatives of free aminogroups or O-acyl derivatives of free hydroxyl-groups and are formed withacyl-groups as for example alcanoyl- or aroyl-groups.

Another object of the present invention are novel OX40R binding agentsconsisting of peptide mimetics (also called peptidomimetics) of thepeptides P5, its specific fragments exemplified by peptides P5-1 andp5-1a, the corresponding active mutants as defined above, in which thenature of peptide or polypeptide has been chemically modified at thelevel of amino acid side chains, amino acid chirality, and/or peptidebackbone. These alterations are intended to provide OX40R binding agentshaving similar (if not improved) therapeutic, diagnostic and/orpharmacokinetic properties.

For example, when the peptide is susceptible to cleavage by peptidasesfollowing injection into the subject is a problem, replacement of aparticularly sensitive peptide bond with a non-cleavable peptide mimeticcan provide a peptide more stable and thus more useful as a therapeutic.Similarly, the replacement of an L-amino acid residue is a standard wayof rendering the peptide less sensitive to proteolysis, and finally moresimilar to organic compounds other than peptides. Also useful areamino-terminal blocking groups such as t-butyloxycarbonyl, acetyl,theyl, succinyl, methoxysuccinyl, suberyl, adipyl, azelayl, dansyl,benzyloxycarbonyl, fluorenylmethoxycarbonyl, methoxyazelayl,methoxyadipyl, methoxysuberyl, and 2,4,-dinitrophenyl. Many othermodifications providing increased potency, prolonged activity, easinessof purification, and/or increased half-life are known in the art (WO02/10195; Villain M et al., 2001). Preferred alternative, “synonymous”groups for amino acids included in peptide mimetics are those defined inTable II.

The techniques for the synthesis and the development of peptidemimetics, as well as non-peptide mimetics, are well known in the art(Sawyer T K, 1997; Hruby V J and Balse P M, 2000; Golebiowski A et al.,2001; Kim H O and Kahn M, 2000). Various methodology for incorporatingunnatural amino acids into proteins, using both in vitro and in vivotranslation systems, to probe and/or improve protein structure andfunction are also disclosed in the literature (Dougherty D A, 2000).

Novel OX40R binding agents can be peptides, peptide mimetics, ornon-peptide mimetics identified by methods of computer-aided drug designwhich make use of the structure and/or sequence of the peptides P5, itsspecific fragments exemplified by peptides P5-1 and P5-1a, or thecorresponding active mutants as defined above. The tridimensionalstructures of OX40L and OX40R, as separate molecules or as a complex,have not been solved yet but the disclosure provided in this patentapplication, once that this information will be available, will allow tostudy the interaction between OX40L and OX40R with greater efficacyusing these and other simulation technologies (Cochran A et al., 2001;Kraemer-Pecore C M et al., 2001). Such computer-assisted analysis can beexploited to develop improved peptide or non-peptide mimetic drugs inthe form of synthetic organic molecules or peptides (for example, havingbetween 4 and 20 amino acid). Once that these compounds have beenscreened and found to be capable of binding OX40R and competing withOX40L, it will then be assessed their utility using cell or animalmodels.

Useful conjugates or complexes of the OX40R binding agents of thepresent invention can be generated, using molecules and methods known inthe art (as shown for anti-OX40R antibodies in WO 95/21251) to improvetheir detection (with radioactive or fluorescent labels, biotin), theirtherapeutic efficacy (with cytotoxic agents), and/or their delivery,with polyethylene glycol and other natural or synthetic polymers (PillaiO and Panchagnula R, 2001).

The peptides P5, its specific fragments exemplified by peptides P5-1 andP5-1a, the corresponding active mutants, and the fusion proteinscontaining them, can be prepared by known chemical synthesis or byrecombinant DNA-based techniques.

Another object of the invention are the nucleic acids encoding for theOX40R binding agents of the invention, including nucleotide sequencessubstantially the same.

“Nucleotide sequences substantially the same” includes all other nucleicacid sequences that, by virtue of the degeneracy of the genetic code,also code for the given amino acid sequences.

The invention also includes vectors of viral or plasmid origin whichallows the expression of the nucleic acid encoding for the OX40R bindingagents of the Invention and prokaryotic or eukaryotic host cellstransformed with such vectors. A stable cell line substantially enrichedin these transformed cells can be isolated, also on the basis of theexpression features of the OX40R binding agent, which can be secreted orexpressed on the membrane surface, for example on human B cells.

OX40R binding agents of the invention can be produced by method whereinthe host cells above described, are cultured in an appropriate culturemedia and the OX40R binding agent is collected.

The DNA sequence coding for the proteins of the invention can beinserted and ligated into a suitable vector. Once formed, the expressionvector is introduced into a suitable host cell, which then expresses thevector(s) to yield the desired protein.

Expression of any of the recombinant proteins of the invention asmentioned herein can be effected in eukaryotic cells (e.g. yeasts,insect or mammalian cells) or prokaryotic cells, using the appropriateexpression vectors. Any method known in the art can be employed.

For example the DNA molecules coding for the proteins obtained by any ofthe above methods are inserted into appropriately constructed expressionvectors by techniques well known in the art. Double stranded cDNA islinked to plasmid vectors by homopolymeric tailing or by restrictionlinking involving the use of synthetic DNA linkers or blunt-endedligation techniques: DNA ligases are used to join the DNA molecules, andundesirable joining is avoided by treatment with alkaline phosphatase.

In order to be capable of expressing the desired protein, an expressionvector should also comprise specific nucleotide sequences containingtranscriptional and translational regulatory information linked to theDNA coding the desired protein in such a way as to permit geneexpression and production of the protein. First in order for the gene tobe transcribed, it must be preceded by a promoter recognizable by RNApolymerase, to which the polymerase binds and thus initiates thetranscription process. There are a variety of such promoters in use,which work with different efficiencies (strong and weak promoters).

For Eukaryotic hosts, different transcriptional and translationalregulatory sequences may be employed, depending on the nature of thehost. They may be derived form viral sources, such as adenovirus, bovinepapilloma virus, Simian virus or the like, where the regulatory signalsare associated with a particular gene which has a high level ofexpression. Examples are the TK promoter of the Herpes virus, the SV40early promoter, the yeast gal4 gene promoter, etc. Transcriptionalinitiation regulatory signals may be selected which allow for repressionand activation, so that expression of the genes can be modulated.

The DNA molecule comprising the nucleotide sequence coding for theprotein of the invention is inserted into vector(s), having the operablylinked transcriptional and translational regulatory signals, which iscapable of integrating the desired gene sequences into the host cell.

The cells that have been stably transformed by the introduced DNA can beselected by also introducing one or more markers allowing for selectionof host cells containing the expression vector. The marker may alsoprovide for phototrophy to an auxotropic host, biocide resistance, e.g.antibiotics, or heavy metals such as copper, or the like. The selectablemarker gene can either be directly linked to the DNA gene sequences tobe expressed, or introduced into the same cell by co-transfection.

Additional elements of the vectors may also be useful for obtaining anoptimal production of proteins of the invention, in particular forselecting a particular cell containing plasmid or viral vector: the easewith which recipient cells, that contain the vector may be recognizedand selected from those recipient cells which do not contain the vector;the number of copies of the vector which are desired in a particularhost; and whether it is desirable to be able to “shuttle” the vectorbetween host cells of different species.

Once the vector(s) or DNA sequence containing the construct(s) has beenprepared for expression the DNA construct(s) may be introduced into anappropriate host cell by any of a variety of suitable means:transformation, transfection, conjugation, protoplast fusion,electroporation, calcium phosphate-precipitation, direct microinjection,etc.

Host cells may be either prokaryotic or eukaryotic. Preferred areeukaryotic hosts, e.g. mammalian cells, such as human, monkey, mouse,and Chinese Hamster Ovary (CHO) cells, because they providepost-translational modifications to protein molecules, including correctfolding or glycosylation at correct sites. Also yeast cells can carryout post-translational peptide modifications including glycosylation. Anumber of recombinant DNA strategies exist which utilize strong promotersequences and high copy number of plasmids that can be utilized forproduction of the desired proteins in yeast. Yeast recognizes leadersequences on cloned mammalian gene products and secretes peptidesbearing leader sequences (i.e., pre-peptides).

After the introduction of the vector(s), the host cells are grown in aselective medium, which selects for the growth of vector-containingcells. Expression of the cloned gene sequence(s) results in theproduction of the desired proteins.

These objects of the invention can be achieved by combining thedisclosure provided by the present patent application on recombinantOX40R binding agents, with the knowledge of common molecular biologytechniques. Many reviews (Makrides S C, 1999) and books providesteachings on how to clone and produce recombinant proteins using vectorsand Prokaryotic or Eukaryotic host cells, such as some titles in theseries “A Practical Approach” published by Oxford University Press (“DNACloning 2: Expression Systems”, 1995; “DNA Cloning 4: MammalianSystems”, 1996; “Protein Expression”, 1999; “Protein PurificationTechniques”, 2001).

Examples of chemical synthesis technologies, which are more indicatedfor producing the OX40R binding agent of the Invention when they are inthe form of peptide or peptide mimetics, are solid phase synthesis andliquid phase synthesis. As a solid phase synthesis, for example, theamino acid corresponding to the C-terminus of the peptide to besynthetized is bound to a support which is insoluble in organicsolvents, and by alternate repetition of reactions, one wherein aminoacids with their amino groups and side chain functional groups protectedwith appropriate protective groups are condensed one by one in orderfrom the C-terminus to the N-terminus, and one where the amino acidsbound to the resin or the protective group of the amino groups of thepeptides are released, the peptide chain is thus extended in thismanner.

Solid phase synthesis methods are largely classified by the tBoc methodand the Fmoc method, depending on the type of protective group used.Typically used protective groups include tBoc (t-butoxycarbonyl), Cl-Z(2-chlorobenzyloxycarbonyl), Br-Z (2-bromobenzyloxycarbonyl), Bzl(benzyl), Fmoc (9-fluorenylmethoxycarbonyl), Mbh(4,4′-dimethoxydibenzhydryl), Mtr(4-methoxy-2,3,6-trimethylbenzenesulphonyl), Trt (trityl), Tos (tosyl),Z (benzyloxycarbonyl) and Cl2-Bzl (2,6-dichlorobenzyl) for the aminogroups; NO2 (nitro) and Pmc (2,2,5,7,8-pentamethylchromane-6-sulphonyl)for the guanidino groups); and tBu (t-butyl) for the hydroxyl groups).After synthesis of the desired peptide, it is subjected to thede-protection reaction and cut out from the solid support. Such peptidecutting reaction may be carried with hydrogen fluoride ortri-fluoromethane sulfonic acid for the Boc method, and with TFA for theFmoc method.

The OX40R binding agents obtained by recombinant DNA or chemicalsynthesis technologies are finally subjected to one or more steps ofpurification. Purification can be carried out by any one of the methodsknown for this purpose, i.e. any conventional procedure involvingextraction, precipitation, chromatography, electrophoresis, or the like.For example, HPLC (High Performance Liquid Chromatography) can be used.The elution can be carried using a water-acetonitrile-based solventcommonly employed for protein purification. The invention includespurified preparations of the OX40R binding agents of the invention.Purified preparations, as used herein, refers to the preparations whichare at least 1%, preferably at least 5%, by dry weight of the compoundsof the invention.

The compounds of the invention described above (proteins, peptides,organic compounds) can be as a medicament, being antagonists of humanOX40L, and in view of the literature on the activity of OX40L as inducerof RANTES expression (Kotani A. et al., 2002), and antagonists of humanRANTES.

The OX40R binding agents of the invention can be used as activeingredient in pharmaceutical compositions for the prophylaxis and/ortreatment of autoimmune diseases, inflammation, or infection.

OX40R binding agent of the invention, once bound to OX40R, acts asantagonist of OX40L, the therapeutical potential of such molecule is inthe prophylaxis and/or treatment of autoimmune diseases (e.g.inflammatory bowel disease, rheumatoid arthritis, and multiplesclerosis), inflammations or infections, where an inhibition of CD4⁺Tcells activation is beneficial. This latter effect can be also used forreducing the population of CD4⁺T cells that express OX40R.

The present invention also provides pharmaceutical compositions for theprophylaxis and/or treatment of diseases related to CD4⁺ T cells,comprising one of the OX40R binding agents of the invention as activeingredient. These pharmaceutical compositions can be formulated incombination with pharmaceutically acceptable carriers, excipients,stabilizers, or diluents. Depending on the properties of the agent, thepharmaceutical composition can be useful for diseases related to CD4⁺ Tcells such as autoimmune diseases, inflammations, or infections.

Pharmaceutical compositions comprising the OX40R binding agents of thepresent invention include all compositions wherein said compound iscontained in therapeutically effective amount, that is, an amounteffective to achieve the medically desirable result in the treatedanimal. The pharmaceutical compositions may contain suitablepharmaceutically acceptable carriers, biologically compatible vehiclessuitable for administration to an animal (for example, physiologicalsaline) and eventually comprising auxiliaries (like excipients,stabilizers or diluents) which facilitate the processing of the activecompounds into preparations which can be used pharmaceutically.

The pharmaceutical compositions may be formulated in any acceptable wayto meet the needs of the mode of administration. The use of biomaterialsand other polymers for drug delivery, as well the different techniquesand models to validate a specific mode of administration, are disclosedin literature (Luo B and Prestwich G D, 2001; Cleland J L et al., 2001).Modifications of the compounds of the invention to improve penetrationof the blood-brain barrier would also be useful. Other methods ofbiomimetic transport and rational drug delivery in the field oftransvascular drug delivery are known in the art (Ranney D F, 2000).

Any accepted mode of administration can be used and determined by thoseskilled in the art. For example, administration may be by variousparenteral routes such as subcutaneous, intravenous, intradermal,intramuscular, intraperitoneal, intranasal, transdermal, oral, or buccalroutes. Parenteral administration can be by bolus injection or bygradual perfusion over time. Preparations for parenteral administrationinclude sterile aqueous or non-aqueous solutions, suspensions, andemulsions, which may contain auxiliary agents or excipients known in theart, and can be prepared according to routine methods. In addition,suspension of the active compounds as appropriate oily injectionsuspensions may be administered. Suitable lipophilic solvents orvehicles include fatty oils, for example, sesame oil, or synthetic fattyacid esters, for example, sesame oil, or synthetic fatty acid esters,for example, ethyl oleate or triglycerides. Aqueous injectionsuspensions that may contain substances increasing the viscosity of thesuspension include, for example, sodium carboxymethyl cellulose,sorbitol, and/or dextran. Optionally, the suspension may also containstabilizers. Pharmaceutical compositions include suitable solutions foradministration by injection, and contain from about 0.01 to 99 percent,preferably from about 20 to 75 percent of active compound together withthe excipient. Compositions which can be administered rectally includesuppositories.

It is understood that the dosage administered will be dependent upon theage, sex, health, and weight of the recipient, kind of concurrenttreatment, if any, frequency of treatment, and the nature of the effectdesired. The dosage will be tailored to the individual subject, as isunderstood and determinable by one of skill in the art. The total doserequired for each treatment may be administered by multiple doses or ina single dose. The pharmaceutical composition of the present inventionmay be administered alone or in conjunction with other therapeuticsdirected to the condition, or directed to other symptoms of thecondition. Usually a daily dosage of active ingredient is comprisedbetween 0.01 to 100 milligrams per kilogram of body weight.

The compounds of the present invention may be administered to thepatient intravenously in a pharmaceutically acceptable carrier such asphysiological saline. Standard methods for intracellular delivery ofpeptides can be used, e.g. delivery via liposomes. Such methods are wellknown to those of ordinary skill in the art. The formulations of thisinvention are useful for parenteral administration, such as intravenous,subcutaneous, intramuscular, and intraperitoneal.

As well known in the medical arts, dosages for any one patient dependsupon many factors, including the patients size, body surface area, age,the particular compound to be administered, sex, time and route ofadministration, general health, and other drugs being administeredconcurrently.

The OX40R binding agents of the present Invention can be used for thedetection of the extracellular domain of OX40R protein, asmembrane-bound or a soluble protein. Obviously this use can be extendedto the detection of activated CD4⁺ T cells expressing OX40R protein.

The method of detections based on these uses comprises a first step inwhich a sample is contacted with the agents or the cells, and a secondstep in which the interaction between the extracellular domain of OX40Rprotein is detected directly (by the means of any label associated tothe agent or the cell, as described before) or indirectly (by the meansof the effects of this binding on the OX40R protein or theOX40R-expressing cell, for example), in order to indicate the presenceof these elements.

The agent or the cells can be immobilized, before or after being put incontact with the sample, onto supports which allow not only thedetection but also the purification, and/or the concentration of theOX40R extracellular domain, as membrane-bound or a soluble protein, orOX40R-expressing cells. These supports can be consequently used inmethods for the detection, the purification, and/or the concentration ofOX40R extracellular domain, as membrane-bound or a soluble protein, orOX40R-expressing cells in a sample by contacting said sample with thesupports, or with cells expressing the OX40R binding agent of thepresent invention. This and the previously described detection methodscan be used to diagnose a condition associated to decreased or increasedpresence of CD4⁺ T cells or of soluble OX40R protein.

he OX40R binding agent of the present invention, or the cells expressingthem, can be administered in methods for the prophylaxis and/ortreatment of autoimmune diseases, inflammations, or infections.

The present invention also discloses screening assay for thedetermination of the nature and the activity of compounds, as providedin Examples of the present patent application, inhibiting OX40R-OX40Linteractions comprising:

a) Forming a sample comprising the following elements:

-   -   i. An element constituting the OX40R binding agent, chosen        amongst the compounds, the cells, and the supports described in        the present invention;    -   ii. An element constituting the OX40R moiety, chosen amongst a        protein comprising the extracellular domain of OX40R, a cell        line expressing OX40R extracellular domain on its surface, and a        cell line secreting extracellular domain of OX40R; and    -   iii. The compound(s) to be tested as inihibitor(s) of        OX40R-OX40L interaction.

b) Detecting, directly or indirectly, the effect of the compounds (iii)on the interactions between the elements (i) and (ii).

c) Comparing the effect detected in (b) amongst samples different interms of quality and/or quantity of the elements of (a).

The support disclosed above represents a preferable element since thosekinds of screening are more efficient and quick by using an bindingelement immobilized on supports like plastic microtiter plate or beads.

The present invention also provides novel kits comprising the OX40Rbinding agents, cells expressing them, or supports comprising them, fordetecting extracellular domain of OX40R protein (as membrane-bound or asoluble protein) or activated CD4⁺ T cells, allowing also the diagnosisof a condition due to a decreased or increased presence of CD4⁺ T cellsor of soluble OX40R protein in a sample obtained from a patient.

Finally, the present invention also provides a kit for screeningcompounds inhibiting the interaction of a protein ligand with amembrane-associated protein, comprising the extracellular portion of themembrane-associated protein and the protein ligand as fusion proteinshaving different tag sequences.

All references cited herein are entirely incorporated by referenceherein, including all data, tables, figures, and text presented in thecited references. Additionally, the entire contents of the referencescited within the references cited herein are also entirely incorporatedby reference. Reference to known method steps, conventional methodsteps, known methods or conventional methods is not in any way anadmission that any aspect, description or embodiment of the presentinvention is disclosed, taught or suggested in the relevant art.

Once understood the features of the methods and products disclosed inpresent application, the necessity and kind of additional steps can beeasily deduced by reviewing prior art, as well as the non-limitingfollowing figures and examples describing the basic details and someapplications of the invention.

EXAMPLES Example 1 Experimental Design for Detecting OX40L-OX40RInteractions Using an AlphaScreen™-Based Competition Assay

Methods

Proteins

Human OX40R-IgG₁ fusion protein has been described previously (Godfreyet al., 1994). The recombinant protein was prepared by constructing amammalian expression vector based on pCEP4 (Invitrogen) in which thecDNA encoding for the extracellular portion of human OX40R (amino acids1-208 of SWISSPROT Acc. N° P43489) was in-frame fused to the 5′ end ofthe cDNA encoding for the constant region of human IgG₁ (hinge region,CH2 and CH3; amino acids 98-330 of SWISSPROT Acc. N° P01857).Recombinant OX40R-IgG₁ is expressed as a secreted protein due to thesignal sequence of OX40R.

HEK293-EBNA cells were transfected, using the calcium phosphatetechnique with the expression construct, which contains a selectablemarker gene for Hygromycin. Cells were seeded at a density of 2×10⁵cells/ml in growth medium (DMEM/F-12 (1:1) supplemented with 10% FetalBovine Serum and 4 mM L-glutamine; Sigma Chemicals). The following day,the medium was replaced by DMEM/F-12 (1:1) supplemented with 2% FetalBovine Serum and 4 mM L-glutamine. One hour later, cells weretransfected and incubated at 37° C. in a humidified atmospherecontaining 5% CO₂ for 4 hours, then medium was changed back to thegrowth medium containing 10% Fetal Bovine Serum. Two days posttransfection, the selection agent (Hygromycin B; 300 μg/ml) was added tothe medium. A clone correctly expressing OX40R-IgG₁ was isolated amongstthe selected cells.

Recombinant OX40R-IgG was purified from the supernatant of a cellculture generated from the isolated clone. The supernatant was clarifiedby centrifugation (10 minutes at 500×g) and subsequently filtered usingPVDF membranes of 0.45 and 0.22 micrometrs pore size (Millipore). Inparallel, the purification column, containing 38 ml resin on whichrecombinant protein G was immobilized (Pharmacia), was equilibrated inloading buffer (0.1 M Tris, pH 7.0) using a Bio-Logic FPLC system(Biorad). After equilibration with 20 column-volumes (CV) of loadingbuffer, the sample was applied to the column at a flow rate of 1 ml/min.The column was washed away with a 10 CV of loading buffer to eliminateprotein bound non-specifically to the resin. OX40R-IgG₁ fusion protein,immobilized on the protein G via the IgG₁ moiety, was eluted by a stepgradient using a 0.1 Glycine/HCl (pH 3.0) elution buffer. The resultingfractions were directly neutralized with 1 M Tris (pH 7.6) to preventprotein degradation in the acidic elution buffer. Finally, the fractionscontaining the OX40R-IgG₁ fusion protein were desalted using a SephadexG25 column (Pharmacia) equilibrated in PBS (Phosphate Buffer Saline) andstored in aliquots at −80° C. until being used for the assays.

Human OX40L-murine CD8 and anti-murine CD8 biotinylated antibodies arecommercially available (Ancell), as well as Suramin (Sigma Chemicals).Recombinant human soluble OX40L was prepared as a fusion protein withGlutathione-S-Transferase (GST) in a Baculovirus Expression System(Gateway™, Invitrogen) by cloning the extracellular portion of OX40Lfused to GST in a plasmid that was then used to transfect SF9 cells.Expression and purification were performed according to Manufacturer'sinstructions.

Alphascreen™ Assay

Alphascreen™ modified acceptor and donor beads were purchased (BiosignalPackard). OX40R-IgG₁ and OX40L-CD8 fusion proteins were immobilized,respectively, onto protein A-conjugated acceptor beads and, by the meansof a biotinylated anti-CD8 antibody, onto streptavidin-conjugated donorbeads.

The binding assay was performed using Costar® 384-well white polystyreneplates (Corning). Each well of a 384-well plate contained a reaction mixhaving a volume of 25 microliters. Each of the five components of thereaction mix (OX40R-IgG₁, OX40L-CD8, biotinylated anti-CD8,streptavidin-conjugated donor beads, and Protein A-conjugated acceptorbeads) was added in a volume of 5 microliters. All dilutions were madein assay buffer (Phosphate Buffer Saline and 0.1% BSA), with or withoutdimethyl sulfoxide (DMSO).

In the standard assay, OX40-IgG₁ and OX40L-CD8 were incubated togetherfor 30 minutes, and then biotinylated anti-CD8 antibody (10 nM) wasadded. Following another incubation period of 30 minutes, streptavidindonor beads (20 μg/ml) and Protein A acceptor beads (20 μg/ml) wereadded to each individual well. The plate was counted 1 hour later usinga Packard Fusion™ reader (Biosignal Packard) set at a read time of 1second/well. Owing to the light sensitivity of the donor and acceptorbeads, the experiments were carried out under blue light and allincubation periods were performed at room temperature. The plate wasread at an excitation wavelength of 680 nanometers combined with ashorter emission wavelength of 520-620 nanometers.

Data Analyses

All KD, IC₅₀ and EC₅₀ values were calculated using Prism® software(Graphpad Software). The transformation of IC₅₀ values into K_(i) valueswas performed using the Cheng-Prusoff equation (Cheng Y C and Prusoff WH, 1973).

Results

OX40L binding assays known in the literature include FACS-based analysis(Taylor L et al, 2002), or a Biacore™-based analysis (Al-Shamkhani A etal., 1997). Being these techniques unsuitable for high throughputscreenings, a more efficient system to establish the potentialinhibiting properties of OX40L-derived peptides on the OX40R-OX40Linteraction was set up by making use of a commercially availabletechnology called Amplified Luminescent Proximity Homogeneous AssayScreen (Alphascreen™; Packard Bioscience), a method based on theLuminescent Oxygen Channeling Immunoassay (LOCI; EP515194; Ullman E etal., 1994).

Briefly, AlphaScreen™ technology provides an easy and reliabledetermination of the effect of compounds on biomolecular interactionsand activities, in particular for protein/protein interaction assays.AlphaScreen™ relies on the use of “Donor” and “Acceptor” polystyrenebeads, each coated with a layer of hydrogel providing functional groupsfor conjugation of a specific molecule. When a biological interactionbetween the immobilized molecules brings the beads into proximity, acascade of chemical reactions is initiated to produce a greatlyamplified signal. Upon laser excitation at 680 nanometers, aphotosensitizer in the “Donor” bead converts ambient oxygen to a moreexcited singlet state. The excited singlet state oxygen moleculesdiffuse across a maximum distance of 200 nanometers before rapidlydecaying. If an acceptor bead is in close proximity, these oxygenmolecules react with a chemiluminescer (such as thioxene derivatives)contained in the acceptor beads to generate chemiluminescence. Theactivated fluorophores subsequently emit light at 520-620 nanometers. Inthe absence of a specific biological interaction, the singlet stateoxygen molecules produced by the donor bead go undetected without theclose proximity of the Acceptor bead. AlphaScreen™ technology allows thedetection of interactions with affinities in thesub-nanomolar/micromolar range.

In the present case, the experimental set-up was first tested using onlythe basic binding partners, immobilized on AlphaScreen™ beads by makinguse of different affinity tags having a short biological linker thatavoids the use of exogenous labeling groups. The signal should bedetected only when streptavidin donor and Protein A acceptor beads areseparated by a distance of less than 200 nm, by virtue of the complexformed by biotinylated anti-CD8 antibody OX40L-CD8, and OX40R-IgG₁ (FIG.1). The IgG₁-tagged extracellular domain of OX40R was incubated withsoluble OX40L-CD8, forming a complex. Owing to the relevant tags, thiscomplex was able to couple to Protein A acceptor beads and, in thepresence of a biotinylated anti-CD8 antibody, and to the streptavidindonor beads, generating an detectable Alphascreen™ signal. In thepresence of a compound competing for the OX40L-OX40R interaction, thedonor and acceptor beads should no longer be in proximity and the signalshould no longer be detected.

In the initial experiments performed to determine the feasibility ofthis assay, the Alphascreen™ signal was increased with the addition ofOX40R-IgG₁ in a dose-dependent manner (FIG. 2A). The calculated EC₅₀values (−logEC₅₀±SEM, Standard Error of the Mean) were 7.7×10⁻⁹ M(8.11±0.04) and 7.9×10⁻⁹ M (8.10±0.01) using 5 or 10 nM OX40-IgG₁.respectively. The experiment was then repeated using a wider range ofreceptor concentrations (2.5-80 nM) in order to determine the optimalconcentration to use for this binding assay. Seven concentrations weretested in total and results demonstrated that each concentrationgenerated a dose-dependent increase of the Signal/Background (S/B) ratioin the Alphascreen™ signal following incubation with OX40L-CD8 (FIG.2B). In all cases, there were no significant differences between thecalculated EC₅₀ values, which remain in the nanomolar range.

Taking these results into account, further experiments were performed ata concentration of OX40R-IgG₁ (10 nM) and of OX40L-CD8 (40 nM) allowinga good S/B window to work with and also ensured that the assay would becost-effective by using only a minimum concentration of these fusionproteins.

The K_(D) of OX40L-CD8 for OX40R-IgG₁ was then calculated by subtractingthe signal due to non-specific binding, as determined in the presence ofsuramin (1 mM), a small molecule inhibiting the interactions TNF-likeproteins with their receptors (Alzani R et al., 1995). Specific bindingof OX40L-CD8 was saturable and of high affinity (FIG. 3), with acalculated K_(D) value of 20.7±5.2 nM. This is in agreement withliterature values when binding affinity of OX40L-CD4 for OX40R wasmeasured using a Biacore sensor chip method (Al Shamkhani A et al.,1997) and, in general, similar to K_(D) values obtained for othermembers of the TNF receptor family for their respective ligands.

The ability of two known competitors of OX40L-OX40R interactions(untagged recombinant human soluble OX40L and suramin), which arethemselves unable to generate an Alphascreen™ signal in this assay inabsence of OX40L-CD8, to displace OX40L-CD8 from OX40R-IgG₁ wasinvestigated. The displacement experiments were performed, taking intoconsideration the approximate K_(D) value, with a concentration ofOX40L-CD8 (40 nM) that was 4 times greater than that of the OX40R-IgG₁(10 nM). Increasing concentrations of OX40L (3 μM-0.1 pM) or suramin (1mM -1 nM) were added to a reaction mixture also containing OX40-IgG₁ (10nM) and OX40L-CD8 (40 nM). Both compounds compete for OX40L-CD8 bindingin a dose-dependent manner, with resulting IC₅₀ values (−logIC₅₀±SEM) of5.9×10⁻⁹ M (8.23±0.35) and 7.9×10⁻⁵ M (4.10±0.06), respectively (FIG.4). These values can be transformed by the Cheng-Prusoff equation(Cheng, Y C and Prusoff, W H, 1973) to K_(i) values of 2.0 nM for OX40Land 26.3 μM for suramin. This latter value for suramin is similar toreported values for inhibition of TNF-α binding to the TNF receptor(Gray P W et al., 1990).

Further optimization of the assay was made to better define theconditions for performing the assay. The assay was able to tolerate,without any loss in signal, a dimethyl sulfoxide (DMSO) concentration upto 1%. The assay can be performed efficiently by combining OX40-IgG₁,OX40L-CD8 and biotinylated anti-CD8 antibody into a first addition step,and combining streptavidin donor and Protein A acceptor beads in asecond addition step, keeping a total incubation time of 60 minutes.Both mixtures can be prepared in advance to addition to the plate.

The Alphascreen™ technology above described, initially developed fordetecting OX40L-derived peptide that competitively inhibited OX40L-OX40Rinteraction, would be suitable to screen also for any other selectivesmall-molecule or peptide inhibitors of such interaction. Since thisassay is homogeneous, highly sensitive, robust and suitable forautomation in a 384-well format, the same approach is potentiallyadaptable to the development of biochemical screens for many otherprotein ligands that interact with membrane-associated proteins, simplyby expressing the extracellular portion of the membrane-associatedprotein and the protein ligand as fusion proteins fusion protein withdifferent tag sequence (IgG₁ and CD8, respectively, in the presentcase).

Example 2 Identification of OX40L-Derived Peptides Binding OX40R

Methods

Peptides

Peptides (10-31 amino acids) were synthesized at purity ranging between85-97% by Epytop (France), and stored in lyophilized form at −20° C. Thepeptides were solubilised in 0.1 mM NaOH in PBS before use. The name,sequence, and the corresponding amino acids in human OX40L for eachpeptide is shown in Table III.

Alphascreen™-Based Competition Assay

The soluble components (OX40-IgG₁, OX40L-CD8 and biotinylated anti-CD8antibody) were mixed at the concentration described before for thecompetition assays with OX40L and suramin (FIG. 4), together withvarious concentrations of each peptide in a 5 microliter volume into afirst addition step. The streptavidin donor and Protein A acceptor beadswere added 30 minutes later. The plates were incubated for 2 hours inthe dark, at room temperature and with shaking, before reading at a longexcitation wavelength of 680 nm combined with a shorter emissionwavelength of 520-620 nanometers, as described in Example 1.

Fluorescence Quenching Assay

The fluorescence quenching assay was performed as previously described(Golabek A et al., 2000). OX40R-IgG₁ (35 micrograms) was dissolved in500 microliters of PBS and the fluorescence spectum was recorded at295-420 nanometers with an excitation wavelength of 290 nanometers usinga spectrofluorimeter (Perkin Elmer LS50B) with the slits set at 5nanometers. Fluorescence spectra of human OX40R-IgG₁ were then recordedin the presence of increasing concentrations (5-1000 nM) of P5 and P5-1peptides after 15 minutes of equilibration. The fluorescence change at336 nanometers was plotted versus peptide concentration and theresulting curves were analyzed by nonlinear regression fit with Prism®software (GraphPad).

Results

A series of partially overlapping peptides were designed on the sequenceof extracellular domain of human OX40L (FIG. 5A; Table III), whichcorresponds to the amino acids 51-183 of the protein. In this area, twopeptides were previously designed on the sequence of mouse OX40L forraising anti-OX40L antibodies (Stuber E and Strober W, 1996).

Using a first series of peptides in the Alphascreen™-based competitionassay described in Example 1, it was possible to define a peptide (P5)capable to inhibit the binding of OX40R to OX40L in the micromolar range(FIG. 5B), an affinity value which is pharmacologically relevant.

On the basis of the sequence of the P5 peptide, a second series ofpartially overlapping peptides were tested to further reduce thisinhibiting molecule (FIG. 6A; Table III). The result of this sequentialscreening is that the region comprised between amino acids 94 and 124 ofhuman OX40L (P5) is not a minimal region since a peptide correspondingto amino acids 107-116 (P5-1), is capable to inhibit the binding ofOX40R to OX40L still in the micromolar range (Kd˜10 and 62 microMolarrespectively). The other tested peptides showed no or hardly measurableeffect on OX40R-OX40L interaction (FIG. 6B).

Since the P4 peptide, which contains the C-terminal six amino acids ofP5-1 peptide, proved to bind OX40R very poorly, it can be also inferredthat the N-terminal amino acids of P5-1 peptide, for example thesequence GYFSQ (peptide P5-1 a; amino acids 107-111 in human OX40L; SEQID NO: 13), may represent a minimal peptide sequence functionally activeas OX40R binding agent. When compared to the mouse OX40L-derived peptideP-OX-1 (Stuber E and Strober W, 1996), this peptide contains twonon-conservative substitutions (FIG. 6A).

The sequences of P5 and P5-1 peptides identified in this example bycompetition assays allowed to identify structures in the OX40L whichplay an essential role in the OX40L-OX40R interaction, demonstratingthat OX40L can be effectively competed by specific peptide sequences.These findings were not predictable from the analysis of the state ofthe art on the structure-activity relationship of these proteins,neither of other TNF/TNFR-like proteins, being the regions of contactbetween proteins belonging to these families very diverse amongst thepairs of ligands and receptors (Bodmer J L et al., 2002).

The affinity of the peptides identified in Example 1 as inhibitor ofOX40L-OX40R interaction was evaluated by fluorescence quenchingspectroscopy, a technology allowing such measurements in solution, undernative conditions and without beads or other supports. This method isbased on monitoring the changes in the intrinsic fluorescence of aprotein (OX40R-IgG₁) upon its binding with another protein (P5 or P5-1).Incubation of OX40R-IgG₁ with increasing concentrations of P5 or P5-1peptides caused a change of its intrinsic fluorescence in the form of anhyperbolic curve, when the changes in OX40R-IgG₁ fluorescence wereplotted against peptide P5 and P5-1 concentration (FIG. 7A and 7B).Non-linear regression analysis of the data revealed an apparentdissociation constant for OX40R of KD˜7.9 nM, and KD˜24.6 nM forpeptides P5 and P5-1, respectively. These values demonstrate a highaffinity interaction between the OX40R-IgG₁ and the selected peptides,which can be therefore used as OX40L antagonists.

The findings presented in this Example indicate that OX40R-OX40Linteraction can be effectively inhibited by using specific OX40L-derivedpeptides which bind efficiently OX40R, providing novel opportunity forthe development of drugs targeting the OX40R pathway and inhibitingaberrant or undesirable physiological events under its control. TheseOX40R binding agents can be then further characterized as antagonist ofOX40L in its interaction with OX40R using the animal and cell biologyassay known in the art (WO 99/42585; Imura A et al., 1997; Nohara C etal., 2001; Pippig S D et al., 1999; Kotani A. et al., 2002), and furthervalidated by testing other potential use-limiting side-effects inrelevant models (Coleman R A et al., 2001). TABLE I Amino More PreferredAcid Synonymous Group Synonymous Groups Ala Gly, Thr, Pro, Ala, Ser Gly,Ala Arg Asn, Lys, Gln, Arg, His Arg, Lys, His Asn Glu, Asn, Asp, GlnAsn, Gln Asp Glu, Asn, Asp, Gln Asp, Glu Cys Ser, Thr, Cys Cys Gln Glu,Asn, Asp, Gln Asn, Gln Glu Glu, Asn, Asp, Gln Asp, Glu Gly Ala, Thr,Pro, Ser, Gly Gly, Ala His Asn, Lys, Gln, Arg, His Arg, Lys, His IlePhe, Ile, Val, Leu, Met Ile, Val, Leu, Met Leu Phe, Ile, Val, Leu, MetIle, Val, Leu, Met Lys Asn, Lys, Gln, Arg, His Arg, Lys, His Met Phe,Ile, Val, Leu, Met Ile, Val, Leu, Met Phe Trp, Phe, Tyr Tyr, Phe ProGly, Ala, Ser, Thr, Pro Pro Ser Gly, Ala, Ser, Thr, Pro Thr, Ser ThrGly, Ala, Ser, Thr, Pro Thr, Ser Trp Trp, Phe, Tyr Trp Tyr Trp, Phe, TyrPhe, Tyr Val Met, Phe, Ile, Leu, Val Met, Ile, Val, Leu

TABLE II Amino Acid Synonymous Group Ser D-Ser, Thr, D-Thr, allo-Thr,Met, D-Met, Met(O), D-Met(O), L-Cys, D-Cys Arg D-Arg, Lys, D-Lys,homo-Arg, D-homo-Arg, Met, Ile, D-.Met, D-Ile, Orn, D-Orn Leu D-Leu,Val, D-Val, AdaA, AdaG, Leu, D-Leu, Met, D-Met Pro D-Pro,L-I-thioazolidine-4-carboxylic acid, D-or L-1-oxazolidine-4-carboxylicacid Thr D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Met(O), D-Met(O), Val,D-Val Ala D-Ala, Gly, Aib, B-Ala, Acp, L-Cys, D-Cys Val D-Val, Leu,D-Leu, Ile, D-Ile, Met, D-Met, AdaA, AdaG Gly Ala, D-Ala, Pro, D-Pro,Aib, .beta.-Ala, Acp Ile D-Ile, Val, D-Val, AdaA, AdaG, Leu, D-Leu, Met,D-Met Phe D-Phe, Tyr, D-Thr, L-Dopa, His, D-His, Trp, D-Trp, Trans-3,4,or 5-phenylproline, AdaA, AdaG, cis-3,4, or 5-phenylproline, Bpa, D-BpaTyr D-Tyr, Phe, D-Phe, L-Dopa, His, D-His Cys D-Cys, S--Me--Cys, Met,D-Met, Thr, D-Thr Gln D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp AsnD-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-Gln Lys D-Lys, Arg, D-Arg,homo-Arg, D-homo-Arg, Met, D-Met, Ile, D-Ile, Orn, D-Orn Asp D-Asp,D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Glu D-Glu, D-Asp, Asp, Asn, D-Asn,Gln, D-Gln Met D-Met, S--Me--Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val

TABLE III Peptide Peptide Correspondance name sequence with human OX40LP1   (SEQ ID NO:2) VASLTYKDKVYLNVTTDNTSLDDFHVNGGEL 150-180 P2   (SEQ IDNO:3) LDDFHVNGGELILIHQNPGEFCVL 160-183 P3   (SEQ ID NO:4)VSHRYPRIQSIKVQFTEYKKEKGFILTSQ  52-80 P4   (SEQ ID NO:5)EKGFILTSQKEDEIMKVQNNSVIINCDGFYL  72-102 P5   (SEQ ID NO:6)IINCDGFYLISLKGYFSQEVNISLHYQKDEE  94-124 P6   (SEQ ID NO:7)HYQKDEEPLFQLKKRSVNSLMVASLTYKDK 118-148 P5-1 (SEQ ID NO:8) GYFSQEVNIS107-116 P5-2 (SEQ ID NO:9) ISLHYQKDEE 107-124 P5-3 (SEQ ID NO:10)GFYLISLKGY  99-108 P5-4 (SEQ ID NO:11) QEVNISLHYQ 111-120 P5-5 (SEQ IDNO:12) IINCDGFYLI  94-103

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1-34. (canceled)
 35. An isolated polypeptide consisting of: a) aminoacids 94-124 (SEQ ID NO: 6) of human OX40L; b) amino acids 94-124 (SEQID NO: 6) wherein one or more amino acids have been deleted; c) aminoacids 107-111 (SEQ ID NO: 13) of human OX40L; d) a peptide sequence ofhuman OX40L having between 5 and 10 amino acids; e) a peptide having thesequence corresponding to 107-116 (SEQ NO ID: 8) or 107-111 (SEQ ID NO:13) of human OX40L; f) an active mutant of a), b), c), d) or e), whereinone or more of the amino acids has been conservatively substituted; g) afusion polypeptide or peptide comprising a), b), c), d), e) or f) and anamino acid sequence belonging to a protein sequence other than humanOX40L (SEQ ID NO: 1); or h) an active fraction, precursor, salt, orderivative of a), b), c), d), e), f) or g).
 36. The isolated polypeptideaccording to claim 35, wherein said fusion polypeptide or peptidecomprises the amino acid sequence belonging to one or more of thefollowing protein sequences: membrane-bound proteins, extracellulardomains of membrane-bound protein, immunoglobulin constant region,multimerization domains, extracellular proteins, signalpeptide-containing proteins, export signal-containing proteins.
 37. Theisolated polypeptide according to claim 35, further comprising amolecule selected from the group consisting of radioactive labels,biotin, fluorescent labels, cytotoxic agents, and drug delivery agents.38. The isolated polypeptide according to claim 35, further comprising asolid support.
 39. An isolated peptide, peptide mimetic, or anon-peptide mimetic designed on the sequence, the structure or thesequence and structure of an amino acid sequence corresponding to107-116 (SEQ NO ID: 8) or 107-111 (SEQ ID NO: 13) of human OX40L. 40.The isolated peptide, peptide mimetic, or a non-peptide mimeticaccording to claim 39, further comprising a molecule selected from thegroup consisting of radioactive labels, biotin, fluorescent labels,cytotoxic agents, and drug delivery agents.
 41. An isolated nucleic acidencoding a polypeptide according to claim
 35. 42. A viral or plasmidvector comprising the nucleic acid of claim
 41. 43. A prokaryotic oreukaryotic host cell which has been transformed with an vector accordingto claim
 42. 44. The prokaryotic or eukaryotic host cell according toclaim 43, wherein said cell is a stable cell line.
 45. The prokaryoticor eukaryotic host cell of claim 43 wherein said cell secretes orexpresses, on the cell surface, a polypeptide consisting of: a) aminoacids 94-124 (SEQ ID NO: 6) of human OX40L; b) amino acids 94-124 (SEQID NO: 6) wherein one or more amino acids have been deleted; c) aminoacids 107-111 (SEQ ID NO: 13) of human OX40L; d) a peptide sequence ofhuman OX40L having between 5 and 10 amino acids; e) a peptide having thesequence corresponding to 107-116 (SEQ NO ID: 8) or 107-111 (SEQ ID NO:13) of human OX40L; f) an active mutant of a), b), c), d) or e), whereinone or more of the amino acids has been conservatively substituted; org) a fusion polypeptide or peptide comprising a), b), c), d), e) or f)and an amino acid sequence belonging to a protein sequence other thanhuman OX40L (SEQ ID NO: 1).
 46. A method of producing a polypeptidecomprising culturing cells of claim 43 and isolating or purifying saidpolypeptide.
 47. A composition comprising a polypeptide according toclaim 35 and a pharmaceutically acceptable carrier, excipient,stabilizer, diluent, or combination thereof.
 48. A screening assay forcompounds modulating OX40R-OX40L interactions comprising: a) forming asample comprising the following elements: i) an element constituting anOX40R binding agent; ii) an element constituting the OX40R moiety,chosen from a protein comprising the extracellular domain of OX40R, acell line expressing OX40R extracellular domain on its surface, or acell line secreting extracellular domain of OX40R; and iii) compound(s)to be tested as modulator(s) OX40R-OX40L interactions; b) detecting,directly or indirectly, the effect of the compounds (iii) on theinteractions between the elements (i) and (ii); and c) comparing theeffect detected in (b) amongst samples different in terms of quality,quantity or both quality and quantity of the elements of (a).
 49. Thescreening assay according to claim 48, wherein said OX40R binding agentis selected from the group consisting of: 1) an isolated polypeptideconsisting of: a) amino acids 94-124 (SEQ ID NO: 6) of human OX40L; b)amino acids 94-124 (SEQ ID NO: 6) wherein one or more amino acids havebeen deleted; c) amino acids 107-111 (SEQ ID NO: 13) of human OX40L; d)a peptide sequence of human OX40L having between 5 and 10 amino acids;e) a peptide having the sequence corresponding to 107-116 (SEQ NO ID: 8)or 107-111 (SEQ ID NO: 13) of human OX40L; f) an active mutant of a),b), c), d) or e), wherein one or more of the amino acids has beenconservatively substituted; g) a fusion polypeptide or peptidecomprising a), b), c), d), e) or f) and an amino acid sequence belongingto a protein sequence other than human OX40L (SEQ ID NO: 1); or h) anactive fraction, precursor, salt, or derivative of a), b), c), d), e),f) or g); 2) an isolated peptide, peptide mimetic, or a non-peptidemimetic designed on the sequence, the structure or the sequence andstructure of an amino acid sequence corresponding to 107-116 (SEQ NO ID:8) or 107-111 (SEQ ID NO: 13) of human OX40L; or 3) a prokaryotic oreukaryotic host secreting or expressing, on the cell surface, apolypeptide consisting of: a) amino acids 94-124 (SEQ ID NO: 6) of humanOX40L; b) amino acids 94-124 (SEQ ID NO: 6) wherein one or more aminoacids have been deleted; c) amino acids 107-111 (SEQ ID NO: 13) of humanOX40L; d) a peptide sequence of human OX40L having between 5 and 10amino acids; e) a peptide having the sequence corresponding to 107-116(SEQ NO ID: 8) or 107-111 (SEQ ID NO: 13) of human OX40L; f) an activemutant of a), b), c), d) or e), wherein one or more of the amino acidshas been conservatively substituted; or g) a fusion polypeptide orpeptide comprising a), b), c), d), e) or f) and an amino acid sequencebelonging to a protein sequence other than human OX40L (SEQ ID NO: 1).50. The screening assay according to claim 49, wherein said polypeptideis attached to a solid support.
 51. A method of treating a disease orcondition comprising the administration of a composition according toclaim 47 to an individual in need of treatment.
 52. A method ofdetecting OX40R comprising contacting a sample with an OX40R bindingagent is selected from the group consisting of: 1) an isolatedpolypeptide consisting of: a) amino acids 94-124 (SEQ ID NO: 6) of humanOX40L; b) amino acids 94-124 (SEQ ID NO: 6) wherein one or more aminoacids have been deleted; c) amino acids 107-111 (SEQ ID NO: 13) of humanOX40L; d) a peptide sequence of human OX40L having between 5 and 10amino acids; e) a peptide having the sequence corresponding to 107-116(SEQ NO ID: 8) or 107-111 (SEQ ID NO: 13) of human OX40L; f) an activemutant of a), b), c), d) or e), wherein one or more of the amino acidshas been conservatively substituted; g) a fusion polypeptide or peptidecomprising a), b), c), d), e) or f) and an amino acid sequence belongingto a protein sequence other than human OX40L (SEQ ID NO: 1); or h) anactive fraction, precursor, salt, or derivative of a), b), c), d), e),f) or g); 2) an isolated peptide, peptide mimetic, or a non-peptidemimetic designed on the sequence, the structure or the sequence andstructure of an amino acid sequence corresponding to 107-116 (SEQ NO ID:8) or 107-111 (SEQ ID NO: 13) of human OX40L; or 3) a prokaryotic oreukaryotic host secreting or expressing, on the cell surface, apolypeptide consisting of: a) amino acids 94-124 (SEQ ID NO: 6) of humanOX40L; b) amino acids 94-124 (SEQ ID NO: 6) wherein one or more aminoacids have been deleted; c) amino acids 107-111 (SEQ ID NO: 13) of humanOX40L; d) a peptide sequence of human OX40L having between 5 and 10amino acids; e) a peptide having the sequence corresponding to 107-116(SEQ NO ID: 8) or 107-111 (SEQ ID NO: 13) of human OX40L; f) an activemutant of a), b), c), d) or e), wherein one or more of the amino acidshas been conservatively substituted; or g) a fusion polypeptide orpeptide comprising a), b), c), d), e) or f) and an amino acid sequencebelonging to a protein sequence other than human OX40L (SEQ ID NO: 1).53. The method according to claim 52, wherein said polypeptide or saidisolated peptide, peptide mimetic, or a non-peptide mimetic is attachedto a solid support.
 54. The method according to claim 53, wherein saidsample contains activated CD4⁺T cells.
 55. The method according to claim53, wherein said sample contains the extracellular domain of OX40Rprotein as membrane-bound or a soluble protein.
 56. A method ofantagonizing the activity of OX40L comprising contacting a compositionaccording to claim 47 with a composition containing OX40R.